Acta Physiol Plant (2015) 37:136

DOI 10.1007/s11738-015-1886-8

ORIGINAL ARTICLE

Ex vitro and in vitro Paphiopedilum delenatii Guillaumin stem

elongation under light-emitting diodes and shoot regeneration
via stem node culture
Vu Quoc Luan1 Nguyen Phuc Huy1 Nguyen Ba Nam1 Trinh Thi Huong1
Vu Thi Hien1 Nguyen Thi Thanh Hien1 Nguyen Thanh Hai1 Do Khac Thinh2

Duong Tan Nhut1

Received: 29 July 2014 / Revised: 4 June 2015 / Accepted: 10 June 2015 / Published online: 1 July 2015
Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2015

Abstract Paphiopedilum spp. are high value horticultural then were maintained under fluorescent light for 60 days.
crops; however, success in Paphiopedilum spp. culture is Although the shoots regenerated new leaves, no new node
still limited. It is therefore necessary to find out adequate was observed. After 180 days of culture, each shoot was
solutions to improve the culture techniques of these cut into five single nodes, and cultured on fern fibers. These
recalcitrant species. In the present study, ex vitro shoots shoots showed to grow vigorously after 12 months.
exposed to LEDs for shoot elongation and in vitro multi-
plication of P. delenatii from single nodes were investi- Keywords Light-emitting diodes
Paphiopedilum
gated. One-month-old ex vitro shoots with 1.52.0 cm delenatii
Node cutting
Darkness
length were grown under various light conditions including
monochromatic blue and red LEDs, mixtures of blue and Abbreviations
red LEDs, and the darkness for investigations of the shoot AC Activated charcoal
elongation. The results indicated that the best stem elon- B5 Gamborg medium
gation was obtained under monochromatic blue LED BA 6-Benzylaminopurine
(100B) after 4 months of culture. The highest shoot IAA Indole-3-acetic acid
regeneration rate (45 %) was also recorded in shoots cul- LED Light-emitting diode
tured on liquid SH medium containing 30 g l-1 sucrose, MS Murashige and skoog medium
1.0 mg l-1 TDZ and 0.3 mg l-1 NAA with cotton wool NAA a-Naphthalene acetic acid
plug as the substrate and exposed to 100B after 45 days. P. PLB Protocorm-like body
delenatii young shoots, which had formed 5 leaves, were SH Schenk and Hildebrandt medium
placed into SH medium supplemented with 0.5 mg l-1 - TDZ Thidiazuron
BA, 0.5 mg l-1 NAA, 30 g l-1 sucrose, and 1 g l-1 acti- VW Vacin and Went medium
vated charcoal in the darkness for stem node elongation
before transferred into greenhouse. After 120 days, the
average shoot length was 10.5 cm, and five new leaves and
five single nodes per shoot were observed. These shoots
Introduction

Communicated by M. Capuana. Paphiopedilum is a beautiful orchid genus, which has high

value in trade as well as ornamental arts. The genus
& Duong Tan Nhut Paphiopedilum has 70 species and is distributed mainly in
duongtannhut@gmail.com
the South Asia and the South East of Asia (Teob 1989).
1
Tay Nguyen Institute for Scientific Research, Vietnam Among Paphiopedilum species, P. delanatii, which is
Academy of Science and Technology, Dalat, Vietnam native to Vietnam, produces delicate pinkish flowers on
2
Institute of Agricultural Science for Southern Vietnam, compact plants (Averyanov et al. 2003). In fact, this orchid
Ho Chi Minh, Vietnam species has a slow growth, and its seeds germinate at low

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rate. To overcome those issues, researchers tried to ger-
minate seeds in vitro and to use plantlets as explants for
axillary shoot induction (Stewart and Button 1975; Arditti
and Ernst 1993; Huang et al. 2001; Chyuam et al. 2010;
Chyuam and Saleh 2011; Patcharawadee et al. 2011).
Recently, Nhut et al. (2005b, 2007) described a wounding
method and stem node elongation of plantlets derived from
seeds germinated in vitro for shoot multiplication. An
efficient shoot multiplication by adopting in vitro cutting
methods was established using in vitro seedlings of
Paphiopedilum Hsinying Rubyweb as the explants source.
This provided a simple means to in vitro propagation of
Paphiopedilum plantlets which produced large numbers of
uniform plantlets in a shorter time compared to the con-
ventional propagation methods (Udomdee et al. 2012).
Propagation from germinated seeds usually generates
the non-homogenous plants compared to the parents. This
is a significant challenge for commercial production
because the separation of colors in the offsprings derived
from seed germination (Sim et al. 2007). Since the first
report of Paphiopedilum micropropagation using young
shoot (Bubeck 1973), the success is still limited. There
were several studies on micropropagation and shoot mul-
tiplication of Paphiopedilum using callus and PLB induc-
tion. However, results indicate that low levels of plant
regeneration were obtained or the tissues did not survive
upon subculture (Lin et al. 2000; Chen et al. 2004; Hong
et al. 2008; Liao et al. 2011).
Light, a primary source of energy, is one of the most
important environmental factors for plant growth (Fukuda
et al. 2008). The intensity and quality of light were proved to
be essential for the growth, morphogenesis and other physi-
ological responses of plants (Rajapakse et al. 1992; Fukuda
et al. 2008; Li and Kubota 2009). Common light sources used
for in vitro plant cultures are fluorescent lamps, metal halide
lamps, high-pressure sodium lamps and incandescent lamps,
which contain unnecessary wavelengths for promoting
growth (Kim et al. 2004). Recently, Light-emitting diodes
(LEDs) have showed to be an alternative light source for
in vitro propagation. LED lighting systems show to have
several advantages, including determined spectral composi-
tion, small size, long life, specific wavelength and minimum
heating (Brown et al. 1995; Bula et al. 1991). The influence of
LEDs on several plants, such as maize (Felker et al. 1995),
strawberry (Nhut et al. 2003), banana (Duong et al. 2003),
potato (Miyashita et al. 1995; Jao and Fang 2004), grape
(Poudel et al. 2008), Cymbidium (Tanaka et al. 1998),
Rehmannia glutinosa (Hahn et al. 2000), Eucalyptus (Nhut
et al. 2002), Lilium (Lian et al. 2002), Chrysanthemum (Hahn
et al. 1998; Kim et al. 2004; Kurilcik et al. 2008),
Spathiphyllum (Nhut et al. 2005a), Zantedeschia (Jao et al.
2005), Euphorbia milii (Dewir et al. 2007), Withania som-
nifera (Lee et al. 2007), and Phalaenopsis orchids (Wongnok
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Acta Physiol Plant (2015) 37:136
et al. 2008), has been reported. Many studies indicate that
LED light is more suitable for plant growth than fluorescent
light. So far, however, there has been little discussion about
utilization of LEDs in cultivation of Paphiopedilum spp.
This is the first study to undertake an application of LED
as a light source to induce shoot elongation of ex vitro P.
delenatii Guillaumin and to improve the regeneration rate
of explants. Those shoots were cultured in the darkness for
120 days to induce stem node elongation and used them as
explant sources for node cutting and further propagation in
the greenhouse.
Materials and methods
Materials
One-month-old Paphiopedilum delenatii Guillaumin
shoots in approximately 1.52.0 cm length derived from ex
vitro grown plants in a greenhouse (under the natural light
no more than 200 lmol m-2 s-1 with sunshade nets, at
1625 C, and 6090 % relative humidity) at the Tay
Nguyen Institute for Scientific Research were used as ini-
tial explant source (Fig. 1a1, a2).
Nutrient media and culture conditions
SH medium (Schenk and Hildebrandt 1972) supplemented
with 1.0 mg l-1 TDZ, 0.3 mg l-1 NAA, 30 g l-1 sucrose
(Luan et al. 2012) and agar (9.0 g l-1) as the solidified
agent or other substrates (gelrite, filter paper, or cotton)
was used as nutrient medium in this study. Medium for
stem node elongation was SH supplemented with
0.5 mg l-1 NAA, 0.5 mg l-1 BA, 30 g l-1 sucrose,
9.0 g l-1 agar, and 1.0 g l-1 activated charcoal (AC). The
pH was adjusted to 5.8 prior to the addition of AC and agar.
After that, media were autoclaved at 121 C, 1 atm for
30 min. Explants were incubated in the darkness, or under
a 16-h photoperiod with light intensity of
1520 lmol m-2 s-1 of cool white fluorescent tubes, or
24-h photoperiod with light intensity of 5 lmol m-2 s-1 of
LEDs depended on experiments.
Explant sterilization
Young shoots with no leaves were cleaned with bleach
(1 % v/v, Sunlight, Unilever Vietnam, HCMC, Vietnam)
and rinsed under tap water for 23 h. After that, the shoots
were immersed in streptomycin (1 mg ml-1) for 30 min
and then washed with 70 % ethanol for 30 s. Then they
were soaked with sterilized water three times. Finally, the
shoots were sterilized with 0.1 % HgCl2 for 6 min and
rinsed with sterilized water four or five times.
Acta Physiol Plant (2015) 37:136 Page 3 of 11 136

Fig. 1 Diagram of P. delenatii

stem elongation and shoot
regeneration via stem node
culture, a1, a2 young ex vitro
shoots with 1.52.0 cm length;
b, c ex vitro elongated shoots
under LED conditions after
4 months; d nodes separated
from young elongation shoot; e1
stem nodes cultured in solid
medium and e2 stem nodes
cultured in liquid medium with
cotton substrate for 1.5 months;
f completely in vitro seedlings
after 3 months; g1 elongated
shoots in the darkness for
4 months; g2 elongated shoots
were transferred to light for
recovering chlorophyll content;
h stem nodes were cut into
orderly positions; i stem nodes
development in greenhouse

Experimental design nodes separately. These nodes then were cultured on SH

medium supplemented with 1.0 mg l-1 TDZ, 0.3 mg l-1
Ex vitro P. delenatii stem node elongation under different NAA, 30 g l-1 sucrose, 9.0 g l-1 agar, and placed under
light conditions fluorescent lights.

The shoots with 1.52.0 cm length derived from ex vitro
plants grown in the greenhouse were transferred into light
chambers with different conditions including 100 % blue
LED (100B, at a wavelength of 450 nm), 100 % red LED
(100R, at a wavelength of 660 nm), 90 % red
LED ? 10 % blue LED (90R:10B, 90 % red light at a
wavelength of 660 nm and 10 % blue light at a wavelength
of 450 nm), 50 % red LED ? 50 % blue LED (50R:50B,
50 % red light at a wavelength of 660 nm and 50 % blue
light at a wavelength of 450 nm) or the darkness to
investigate effect of light conditions on shoot elongation
(Fig. 1b). Cultures with LED treatments were maintained
at 1625 C (day and night), with 30 lmol m-2 s-1 pho-
tosynthetic photon flux (PPF) under a 24-h photoperiod.
After 4 months, in vitro young shoots with five leaves were
used as explants in node cutting experiments (Fig. 1c, d).
By this way, contamination of fungi and bacteria from
substrates could be limited and the explants were harvested
easily.
Regeneration ability of explants derived from young shoot
under different light conditions
Effect of medium substrates on node stems regeneration
The best elongated ex vitro 4-month-old shoots derived
from node cuttings under LED in previous treatment were
sterilized and cut into nodes before cultured on SH medium
supplemented with 1.0 mg l-1 TDZ, 0.3 mg l-1 NAA with
different substrates including cotton, filter papers, gelrite or
agar (Fig. 1e1, e2). All explants were placed under fluo-
rescent lights.
Effect of different nutrient media on in vitro P. delenatii
shoots growth and development
The in vitro shoots harvested from previous treatments
were cultured on different nutrient media such as MS
(Murashige and Skoog 1962); MS; VW (Vacin and Went
1949); B5 (Gamborg et al. 1968) and SH supplemented
with 0.5 mg l-1 BA, 0.5 mg l-1 NAA (Nhut et al. 2007),
9.0 g l-1 agar, 30 g l-1 sucrose, and 1.0 g l-1 AC, and
then set under fluorescent lights (Fig. 1f).
In vitro P. delenatii shoot elongation in the darkness

Ex vitro 4-month-old shoots under different light condi- To investigate effect of the darkness on shoot elongation,
tions in previous experiment were sterilized and cut into 5-leaf young shoots were cultured on SH medium

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supplemented with 0.5 mg l-1 NAA, 0.5 mg l-1 BA,
30 g l-1 sucrose, 9.0 g l-1 agar, and 1.0 g l-1 AC, and
placed in the darkness without subculture (Fig. 1g1). The
shoot length was measured after 14 months of culture.
Stem node survival in green house
The 4-month-old in vitro shoots placed in the darkness
were transferred into fluorescent light condition at
1520 lmol m-2 s-1 and regime of 16/8-h (light/dark)
(Fig. 1g2). Temperature was maintained at 25 3 C.
After 2 months, these shoots were cut into single nodes and
cultivated in fern fibers in the greenhouse (under the nat-
ural light with PPFD less than 200 lmol m-2 s-1 using
sunshade nets, at 1625 C, and 6090 % relative humid- and Ernst 1992). We assume that the different light regimes
ity) for 12 months (Fig. 1h, i). SPAD values of the shoots
and single nodes were determined by the Minolta Chloro-
phyll meter SPAD-502 (Minolta Co., Ltd, Osaka 541,
Japan) after 4 months in the darkness, 2 months under
fluorescent lamp and 12 months in the green house.
Statistical analysis
In ex vitro stem node elongation experiment, each treat-
ment was applied in five pots with three young shoots per
pot. Shoot regeneration was obtained from 20 samples. In
in vitro shoot elongation experiments, each treatment was
applied in five culture bottles, and each bottle contained
five shoots. In node cutting experiments and growing in
green house, each treatment was applied with 25 nodes,
and 5 nodes were cultivated in one pot. All experiments
were repeated 4 times. Data were analyzed by SPSS 16.0
software with Duncans multiple range test p value B0.05
(Duncan 1995).
Results and discussion
Ex vitro stem node elongation under various light
conditions
Table 1 provides the summary statistics for effects of LED
on elongation of P. delenatii stem nodes after 4 months.
There is a significant difference on length among the young
shoots placed under various light conditions (Fig. 4af).
The most striking observation to emerge from the data
comparison was the longest shoots obtained under 100B.
Those shoots have node separation visually, where the
emergent leaves are same size in both length and width.
Particularly, the shoot meristem promoted stem elongation
rather than number of nodes increases (Fig. 4e).
The results showed that each node consists of one leaf.
Nodes cultured under 100B induced leaf formation (5.7
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Acta Physiol Plant (2015) 37:136
leaves/shoot) significantly higher than those (4.755
leaves/shoot) placed under other light conditions (Table 1).
Stem elongation of dark-grown shoots also was observed
with an average shoot length of 8.87 cm. Upper nodes with
immature, soft, small and light yellow leaves were
observed (Fig. 4f). Following the shoots in the treatment
with 100B and the darkness, the shoots grown under 100R
or combination of red LED and blue LED resulted in better
stem elongation compared with the control (Table 1).
However, Duncans test did not show significant differ-
ences, and these shoots were not suitable for node cutting
and regeneration (Fig. 4bd). These results may be
explained by the effect of light wave length, concentration
and photoperiod on plant growth and development (Arditti
have affected phytochrome B (phyB) response to varying
degrees. In the darkness, of course, there was no phyB
activation (Parks et al. 2001; Platten et al. 2005). Indeed,
photoperiod impacts on different species in different ways,
some crops (Malus cultivars, Fuji apple, Strelitzia regi-
nae, etc.) response well in the darkness while some
(Jonathan apple, Malus prunifolia, etc.) in continuous
light and others (Rosa hybrida, Columar apple trees, etc.)
under intermediate photoperiod (Ziv and Halevy 1983;
Yepes and Aldwinckle 1994; Wang et al. 1994; Noiton
et al. 1992; Schaefer et al. 2002; Marin et al. 1993; Bressan
et al. 1982). In addition, the light intensity also affects
shoot quality, chlorophyll content, and leaf length (Morini
et al. 1991). In the current study, the best shoot elongation
was observed under 100B. This seems to be contradicting
previous studies on tomato where red light promoted
flowering and blue light suppressed stem elongation
(Giliberto et al. 2005; Hirai et al. 2006). Conversely,
according to Nanya et al. (2012) stem elongation in tomato
seedlings depended on the quantity of blue light. A possible
explanation for this might be that blue LED at low intensity
(5 lmol m-2 s-1) promoted the shoot elongation of P.
delenatii in this study.
Regeneration ability of explants derived from young
shoots under different light conditions
The success of Paphiopedilum micropropagation utilizing
ex vitro-derived explants has been relatively limited due to
not only the rarity of materials but also the bacterial and
fungal decontamination of ex vitro-derived explants and
the poor development of explants that survive under
in vitro conditions (Stewart and Button 1975; Huang 1988).
Indeed, Paphiopedilum species and hybrids remain the
only commercially grown orchids that are not cloned
(Huang 1988; Liao et al. 2011; Ng and Saleh 2011). So far,
there have been only three reports of Paphiopedilum
micropropagation from ex vitro-derived explants (Stewart
Acta Physiol Plant (2015) 37:136 Page 5 of 11 136

Table 1 Effect of light conditions on ex vitro P. delenatii stem node elongation

Light conditions Average height (cm) Number of leaves/shoot Average leaf length (cm) Average leaf width (cm)

Control (in greenhouse) 2.25e 4.75b 9.35a 3.72a

d b b
50R:50B 4.37 4.75 6.05 1.62b
c ab b
90R:10B 7.30 5.00 5.92 1.60b
100R 4.97d 4.75b 5.97b 1.70b
a a c
100B 11.00 5.75 5.47 1.35c
b ab b
Darkness 8.87 5.00 6.12 1.60b
Different letters in the same column indicate significant difference in Duncans test (p value B0.05)

same regeneration rateat low level18.75 % together

Fig. 2 Effect of explant sources under various light conditions on
survival, necrosis, and contamination rates after 1.5 months of culture
with a high contamination rate of 52.50 % (Fig. 2).
Although the shoots grown in the darkness were found to
give a significant stem node elongation, the low survival
rate of 10.00 % was recorded. In this experiment, the
shoots grown under 100B gave the highest regeneration
rate (33.50 %) (Fig. 4g). According to Tibbitts et al.
(1983), blue light is important in chlorophyll biosynthesis,
stomata opening, enzyme synthesis, maturation of chloro-
plasts and photosynthesis. The blue light is also indis-
pensable for the morphologically healthy plant growth
(Giedre et al. 2010). Therefore, shoots were pre-treated
under 100B might have a higher vitality comparing to the
others.

and Button 1975; Huang 1988; Liao et al. 2011). Stewart
and Button (1975) used young and mature flower stems,
tips of leaves and roots, stamens, ovaries and terminal buds
of P. villosum, P. fairrieanum and P. insigne to induce
callus. Among 126 explants, only 29 (23.02 %) were not
contaminated and only six remaining explants induced
callus formation.
In the present study, ex vitro 4-month shoots grown
under different light conditions in the previous experiment
were used as initial explants for regeneration. After ster-
ilization, the shoots were cut into single nodes and these
nodes then were cultured into culture vessels. After
45 days of culture, the results indicated that the young
shoots grown under different light conditions gave a sig-
nificantly higher regeneration rate compared with the
control except shoots placed under 50R:50B. Interestingly,
what is surprising in the current study is that lower con-
tamination rates were recorded (37.5052.50 %) when
compared with previous studies.
Plants exposed to 100R and 90R:10B revealed signifi-
cantly higher regeneration and lower contamination rates
compared to the control (Fig. 2). It might be explained that
under these conditions the stem elongation was better than
the control, and single nodes were obtained. These nodes
then were effectively surface sterilized. In contrast, the
shoots grown under 50R:50B and the control resulted in the
Effect of substrates on regeneration of P. delenatii
stem nodes
The shoots obtained from 100B treatment were used as
explants for sterilization. The shoots were cut into single
nodes and cultured onto different substrates in order to
investigate the survival and regeneration rates. In liquid
culture, uptake of nutrients and plant growth regulators is
more effective than in solid culture (Ziv 1989; Smith and
Spomer 1994; Sandal et al. 2001), therefore, shoots and
roots cultured in liquid medium usually gave better growth
and development. In this study, there were significant dif-
ferences of regeneration rate among nodes cultured with
various substrates after 45 days. The highest regeneration
rate (45.00 %) was observed in the treatment using cotton
as the substrate (Fig. 4h). There was no significant differ-
ence in regeneration rate between nodes on other types of
substrates including filter paper, gelrite and agar (Fig. 3).
Effect of nutrient media on growth and development
of in vitro P. delenatii shoots
The results indicated that nutrient medium affected shoot
growth and development after 90 days of culture. Since the
sixtieth day of culture, the young leaves turned light white
in color when shoots cultured on MS medium, then they

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Fig. 3 Effect of substrates on ex vitro P. delenatii shoot regeneration
after 1.5 months of culture
wilted gradually and became necrotic after 90 days of
culture (Fig. 4i5). This result was in accordance with the
study of Luan et al. (2012), when Paphiopedilum Van
Hai shoots died after 90 days of culture on MS medium.
The shoots cultured on MS also were found to grow
slowly with abnormalities. Leaves of these shoots were
light green gradually since the sixtieth day of culture and
turned yellow brown and became necrotic by the ninetieth
day (Fig. 4i4).
The wilting of shoots might be caused by the high
NH4NO3 concentration in MS or MS. It had also been
reported that a high ratio of NH4?:NO3- increase number
of wilt leaves (Barker et al. 1987). It could be possible that
high ratio of NH4?:NO3- in medium might cause leaves
wilting due to that high NH4? concentration induced
ethylene production subsequently caused shoot senescence.
In addition, NH4? and NO3- play important roles in pH
balance of medium that controls essential elements
absorption in plants (Ramage and Williams 2001). Plantlets
developed well in VW and B5 nutrient media (Fig. 4i3, i2).
Leaf length and fresh weight were significantly different
between shoots on B5 and VW media (Table 2). The best
P. delenatii growth and development were observed on
medium SH (Fig. 4i1), (Table 2).
Shoot elongation of in vitro P. delenatii
in the darkness
A number of studies have examined various factors
impacting on stem growth and elongation in dark-grown
plants (Parker et al. 1949; Cohen et al. 1991; Behringer and
Davies 1992; Sorce et al. 2008; Tomoki et al. 2014). The
studies reported that stem elongation in dark-grown plants
is controlled by IAA, BA, a novel triazole derivative with
coumarin moiety (YCZ-FL) or the phytochrome system.
However, previous studies have not dealt with stem elon-
gation of in vitro Paphiopedilum. In the current study, total
darkness was exploited for in vitro shoot elongation.
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Acta Physiol Plant (2015) 37:136
Table 3 presents the effect of the darkness on shoot elon-
gation after 120 days of culture. Young shoots cultured in
the darkness showed a significant difference in height after
30, 60, 90, and 120 days (Table 3). In this study, after
30 days of culture, the first node initially elongated and
young green leaves formed. After 60 days of culture, shoot
length increased significantly. Internodes at position num-
ber 2 (from top) elongated; however, there was no root
formation at nodes. Observation after 90 days of culture
showed that shoot length reached 7.12 cm, stem internode
length was 1.01.5 cm, and the leaf length and width were
2.22 and 1.15 cm, respectively (Table 3). Root formation
was also observed in these shoots. After 120 days of cul-
ture in the darkness, the shoot height was 10.50 cm with 5
new leaves though the leaves did not grow considerably
(Fig. 4j) while new roots formed with the average number
of roots per shoot of 3.37. Previous report demonstrated
that using LED promoted shoot elongation in P. delenatii,
and the height can reach up to 10 cm under 100R and low
intensity of fluorescence (Nhut et al. 2007). In this study,
the shoot length was observed at 10.50 cm with five leaves
per shoot after 120 days of culture in the darkness.
Survival rate of different node positions after
12-month transferred into the greenhouse
A number of researchers reported the propagation of orchid
species, including Phalaenopsis, Dendrobium, Rhyn-
chostylis, etc., through in vitro culture of flower stalk and
stem nodes (Rotor 1949; Urata and Iwanaga 1965; Arditti
et al. 1973; Mosich et al. 1973, 1974a, b; Koch 1974;
Nuraini and Shaib 1992; Chen and Pileuk 1995; Le et al.
1996). However, far too little attention has been paid to
controlling light quality in order to enhance the multipli-
cation rate of Paphiopedilum, a recalcitrant orchid species,
through stem node culture. It has been demonstrated that
the moderate PPF of 30 lmol m-2 s-1 is effective for
vigorous and long stem with separating internodes. The
nodes were then cut into nodal segment and sub-cultured
onto MS modified medium, and the stem node-derived
healthy plantlets grew and developed well in green house
(Nhut et al. 2007). In the present study, in vitro shoots were
elongated in the darkness and transferred into light (under
fluorescent lamps). Those shoots then were cut into single
nodes (Fig. 4k) and grown on Taiwan fern fibers. Several
weeks after being transferred into ex vitro conditions, most
of the nodes (with one or more roots before being cut
separately) showed to grow and develop well while those
without any roots in the same time resulted in the necrosis.
After 12-month culture, there were significant differences
in growth and development as well as survival rates from
various stem nodes. All position-1 shoots (corresponding to
top nodes) survived (100 %). In addition, there was an
Acta Physiol Plant (2015) 37:136
Fig. 4 Micropropagation of Paphiopedilum delenatii via ex vitro and
in vitro stem node elongation. a Control sample in the greenhouse;
b shoot growth under 50R:50B; c shoot growth under 90R:10B;
d shoot growth under 100R; e shoot growth under 100B; f Shoot
growth in the darkness; g young shoots derived from 100B-treated
shoots after 45 days culture on agar medium; h young shoots derived
from 100B-treated shoots after 45 days culture on cotton substrates;
increase in other parameters examined (Table 4),
(Fig. 4m). The high survival rate could be explained since
those shoots regenerated 3 leaves and 2 shoots in average
which may have contributed to enhance photosynthesis and
water absorption of shoots (Fig. 4k). However, the second
Page 7 of 11 136
i shoot growth in various nutrient media: 1 SH, 2 B5, 3 VW, 4 MS,
5 MS after 90 days of culture; j in vitro shoot elongation in dark after
120 days culture; k node cuttings and direct culture on Taiwan fern
fibers; l plantlets formation from stem nodes after 12 months culture
in the greenhouse (numbers 15 corresponding to position of stem
node from top to bottom); m healthy plantlets derived from node
position-1 grown in the greenhouse after 12 months
stem node showed the lowest survival rate (23.75 %).
There was no lateral shoot formation in those shoots. It is
possible that those stem nodes were still very young and
easily infected by pathogens while growing in the
greenhouse.
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Table 2 Effect of nutrient

Nutrient Leaf length/ Leaf Root number/ Fresh weight/ Morphological
medium on P. delenatii shoot
medium shoot (cm) number/shoot shoot shoot (g) features/phenotype
growth and development
MS 1.75d 2.95d 1.47d 0.50e Brown, die
c c c
MS 3.87 3.66 2.60 1.07d Light green, young leaf
VW 4.50b 3.95bc 3.42b 1.35c Green leaves, stubborn
a b b b
B5 5.00 4.15 3.37 1.55 Green
SH 5.27a 4.57a 3.95a 1.195a Green
Different letters in the same column indicate significant difference in Duncans test (p value B0.05)

Table 3 Effect of dark regime on in vitro P. delenatii shoot elongation

Dark regime Shoot New New leaf New leaf Root Leaf phenotype
(day) height (cm) leaves/ shoot length (cm) width (cm) number/
node

Control 120 days of light 4.25d 3.00c 4.95a 3.37a 0.00c Dark green shoots
d e b b c
30 4.37 1.00 3.80 1.65 0.00 Young leaf, light green
60 5.65c 2.42d 3.00c 1.45bc 0.00c Yellow latter leaf, white green young leaf
90 7.12b 3.75b 2.22d 1.15c 1.82b Yellow lower leaf, white green upper leaf
120 10.50a 5.00a 2.00d 1.12c 3.37a Brown lower leaf, white green upper leaf
Different letters in the same column indicate significant difference in Duncans test (p value = 0.05)

Table 4 Effect of node position on survival rate and development in the greenhouse after 12 months
Node SPAD value SPAD value after SPAD value after Survival New New Average Shoot phenotype
position after 2 months 12 months (%) shoot/ leaves leaf
4 months in the transferred transferred sample width (cm)
darkness into fluorescence into green house

1 3.81e 47.00a 44.68a 100.00a 1.00a 3.87a 3.07a Big shoot, healthy
d b c d d d
2 5.15 44.46 0.000 23.75 0.00 0.00 0.00d No shoot formation
3 9.93b 42.55b 41.36b 33.75c 0.75b 2.50c 1.42c Weak, light green
4 13.73a 36.33b 39.30b 32.50c 0.72b 3.12b 1.45c Weak, light green
5 9.20c 33.10c 40.93b 56.25b 0.53c 4.12a 2.12b Big shoot, dark
green
Different letters in the same column indicate significant difference in Duncans test (p value = 0.05)

Similar to the second stem node, the third and fourth
ones were found with low survival rates, 33.75 and
32.50 %, respectively. However, among the survived
explants from the stem nodes 3 and 4, there were lateral
shoot formation -0.75 and 0.72 shoot/explant, respec-
tively (Table 4), and new leaf numbers were 2.50 and
3.12 leaves/shoot, respectively (Fig. 4l). The stem node
position number 5, corresponding to bottom node, gave
the higher survival rate (56.25 %) but lower number of
new shoots (0.53 shoot/explant) (Table 4). The node
cutting method and regeneration of whole in vitro P.
delenatii plant were described by Nhut et al. (2007), in
which 75 % of new plants were regenerated. In this study,
the top shoots showed a survival rate of 100 % and three
intact plants could be obtained from one in vitro single
plantlet.
Conclusions
The shoots grown under 100 % blue LED showed the
highest shoot elongation (11 cm) and number of nodes
(5.75 nodes/shoot). The regeneration rate reached the
highest (45 %) under 100 % blue LED treatment with
cotton used as the substrate in culture. SH medium sup-
plemented with 0.5 mg l-1 NAA, 0.5 mg l-1 BA, 30 g l-1
sucrose, 9.0 g l-1 agar, and 1.0 g l-1 activated charcoal
(AC) was suitable for plant growth and development.
Shoots cultured in the darkness for 120 days showed the
best stem elongation (10.5 cm) and number of nodes (5
nodes/shoot). Those regenerated shoots (from single nodes)
were cultured under fluorescent lights for 60 days with
intensity of 1520 lmol m-2 s-1 and a regime of 16/8 h
(light/dark). Survival and growth rate were optimal with

123
Acta Physiol Plant (2015) 37:136
fern fiber substrate in the greenhouse. Consequently, the
node 1 generated an average number of leaves per shoot of
3.87 with the leaf width of 3.07 cm. The results suggested
that in vitro shoots initially treated with LED for elongation
after 180 days can be cut into 3 single nodes and then
cultured on fern fiber in the greenhouse. Ultimately, those
nodes will develop into healthy plants after 12 months
under ex vitro conditions.
Author contribution statement Vu Quoc Luan designed
and carried out the experiments for ex vitro P. delenatii
Guillaumin stem elongation under light-emitting diodes
and shoot regeneration via stem node culture in vitro,
Nguyen Phuc Huy, Vu Thi Hien, Trinh Thi Huong and Do
Khac Thinh assisted in carrying out the experiments while
Nguyen Ba Nam designed the LEDs system. Duong Tan
Nhut, Nguyen Phuc Huy, Nguyen Thi Thanh Hien and
Nguyen Thanh Hai were responsible for verification of the
paper.
Acknowledgments This work was financially supported by the
National Foundation for Science and Technology Development
(NAFOSTED), Vietnam, under Project No 106.16-2012.32.
References
Arditti J, Ernst R (1992) Micropropagation of orchids. John Wiley
Sons Inc, Hoboken, pp 434466
Arditti J, Ernst R (1993) Several early scientific investigators and
their research: A contribution towards the history of orchids in
the United States of America. In: Rittershausen W (ed) 100 years
of orchids, the Orchid review centenary year book 18931993.
Bradfield Books, Creat Bardfliled, Braintree, Essex CM7 4RZ,
UK, pp 125142
Arditti J, Mosich SK, Ball EA (1973) Dendrobium node cultures: a
new means of clonal propagation. Aust Orchid Rev 38:175179
Averyanov L, Cribb P, Loc PK, Hiep NT (2003) Slipper orchids of
Vietnam. Compass Press Limited, Kew
Barker AV, Arker K, Corey A (1987) Ammonium-induced ethylene
evolution by horticultural crops. Hort Sci 22:381
Behringer FJ, Davies PJ (1992) Indole-3-acetic acid levels after
phytochrome-mediated changes in the stem elongation rate of
dark- and light-grown Pisum seedlings. Planta 188:8592
Bressan PH, Kim YJ, Hyndman SE, Hasegawa PM, Bressan RA
(1982) Factors affecting in vitro propagation of rose. J Am Soc
Hortic Sci 107:979990
Brown CS, Schuerger AC, Sager JC (1995) Growth and photomor-
phogenesis of pepper plants under red light-emitting diodes with
supplemental blue or far-red lighting. J Amer Soc Horti Sci
120:808813
Bubeck SK (1973) A study of Paphiopedilum meristem culture. Ph.D
Thesis, Rutgers University, University Microfilm International,
Ann Arbor, Michigan
Bula RJ, Morrow RC, Tibbitts TW, Ignatius RW, Martin TS, Barta DJ
(1991) Light emitting diodes as a radiation source for plants.
HortScience 26:203205
Chen Y, Pileuk C (1995) Effects of thidiazuron and N6-benzy-
laminopurine on shoot regeneration of Phalaenopsis. Plant
Growth Regul 16:99101
Page 9 of 11 136
Chen TY, Chen JT, Chang WC (2004) Plant regeneration through
direct shoot bud formation from leaf cultures of Paphiopedilum
orchids. Plant Cell Tiss Org Cult 76:1115
Chyuam YN, Saleh NM (2011) In vitro propagation of Paphiope-
dilum orchid through formation of protocorm-like bodies. Plant
Cell Tiss Org Cult 105:193202
Chyuam YN, Saleh NM, Zaman FQ (2010) In vitro multiplication of
the rare and endangered slipper orchid, Paphiopedilum roth-
schildianum (Orchidaceae). Afr J Biotech 9(14):20622068
Cohen L, Gepstein S, Horwitz BA (1991) Similarity between
cytokinin and blue light inhibition of Cucumber hypocotyl
elongation. Plant Physiol 95(1):7781
Dewir YH, Chakrabarty D, Hahn EJ, Paek KY (2007) Flowering of
Euphorbia millii plantlets in vitro as affected by paclobutrazol,
light emitting diodes (LEDs) and sucrose. Acta Hort
764:169173
Duncan DB (1995) Multiple range and multiple F test. Biometrics
11:142
Duong TN, Hong LTA, Watanabe H, Goi M, Tanaka M (2003)
Efficiency of a novel culture system by using light-emitting
diode (LED) on in vitro and subsequent growth of microprop-
agated banana plantlets. Acta Hort 616:121127
Felker FC, Doehlert DC, Eskins K (1995) Effects of red and blue light
on the composition and morphology of maize kernels grown
in vitro. Plant Cell Tiss Org Cult 42:147152
Fukuda N, Fujitan M, Ohta Y, Sase S, Nishimura S, Ezura H (2008)
Directional blue light irradiation triggers epidermal cell elonga-
tion of abaxial side resulting in inhibition of leaf epinasty in
geranium under red light condition. Sci Hort 115:176182
Gamborg O, Miller R, Ojima K (1968) Nutrient requirement
suspensions cultures of soybean root cells. Exp Cell Res
50(1):151158
Giedre S, Ausra B, Akvile U, Gintare S, Pavelas D (2010) The effect
of red and blue light component on the growth and development
of frigo strawberries. Zemdirbyste Agr 97(2):99104
Giliberto L, Perrotta G, Pallara P, Weller JL, Fraser PD, Bramley PM,
Fiore A, Tavazza M, Giuliano G (2005) Manipulation of the blue
light photoreceptor cryptochrome 2 in tomato affects vegetative
development, flowering time, and fruit antioxidant content. Plant
Physiol 137:199208
Hahn EJ, Bae CH, Lee YB (1998) Growth and leaf-surface
characteristics of Chrysanthemum plantlets between microprop-
agation and microponic system. J Kor Soc Hort Sci 39:838842
Hahn EJ, Kozai T, Paek KY (2000) Blue and red light-emitting diodes
with or without sucrose and ventilation affects in vitro growth of
Rehmannia glutinosa plantlets. Plant Biol 43:247250
Hirai T, Amaki W, Watanabe H (2006) Action of blue or red
monochromatic light on stem intermodal growth depends on
plant species. Acta Hort 711:345349
Hong PI, Chen JT, Chang WC (2008) Plant regeneration via
protocorm-like body formation and shoot multiplication from
seed derived callus of a maudiae type slipper orchid. Acta
Physiol Plant 30:755759
Huang LC (1988) A procedure for asexual multiplication of
Paphiopedilum in vitro. Am Orchid Soc Bul 57:274278
Huang LC, Lin CJ, Kou CI, Huang BL, Murashige T (2001)
Paphiopedilum cloning in vitro. Sci Hort 91:111121
Jao RC, Fang W (2004) Effects of frequency and duty ratio on the
growth of potato plantlets in vitro using light-emitting diodes.
HortScience 39:375379
Jao RC, Lai CC, Fang W, Chang SF (2005) Effects of red light on the
growth of Zantedeschia plantlets in vitro and tuber formation
using light-emitting diodes. HortScience 40:436438
Kim SJ, Hahn EJ, Heo JW, Paek KY (2004) Effects of LEDs on net
photosynthetic rate, growth and leaf stomata of Chrysanthemum
plantlets in vitro. Sci Hort 101:143151
123
136 Page 10 of 11
Koch L (1974) Erbgleiche Vermehrung von Phalaenopsis in vitro.
Gartenwelt 74:482484
Kurilcik A, Miklusyte-Canova R, Dapkunien_e S, Zilinskait_e S,
Kurilcik G, Tamulaitis G, Duchovskis P, Zukauskas A (2008)
In vitro culture of Chrysanthemum plantlets using light-emitting
diodes. Cent Eur J Biol 3:161167
Le BV, Hong Phuong NT, Anh Hong LT, Van Tran Thanh K (1996)
High frequency shoot regeneration from Rhynchostylis gigantea
(Orchidaceae) using thin cell layer. Plant Growth Regul
28:179185
Lee AE, Tewari RK, Hahn EJ, Paek KY (2007) Photon flux density
and light quality induce changes in growth, stomatal develop-
ment, photosynthesis and transpiration of Withania somnifera
(L.) Dunal. plantlets. Plant Cell Tiss Org Cult 90:141151
Li Q, Kubota C (2009) Effects of supplemental light quality on
growth and phytochemicals of baby leaf lettuce. J Environ Exp
Bot 67:5964
Lian ML, Murthy HN, Paek KY (2002) Effect of light emitting diodes
(LEDs) on the in vitro induction and growth of bulblets of Lilium
oriental hybridPesaro. Sci Hort 94:365370
Liao YJ, Tsai YC, Sun YW, Lin RS, Wu FS (2011) In vitro shoot
induction and plant regeneration from flower buds in Paphio-
pedilum orchids. In Vitro Cell Dev Biol Plant 47:702709
Lin YH, Chang C, Chang WC (2000) Plant regeneration from callus
culture of a Paphiopedilum hybrid. Plant Cell Tiss Org Cult
62:2125
Luan VQ, Huy NP, Chien HX, Huong TT, Hien VT, Nam NB, Thinh
DK, Nhut DT (2012) Micropopagation of Paphiopedilum
callosum. J Biotech 10(3):487494
Marin JA, Jones OP, Hadlow WCC (1993) Micropropagation of
columnar apple trees. J Hortic Sci 68:289297
Miyashita Y, Kitaya Y, Kozai T, Kimura T (1995) Effects of red and
far-red light on the growth and morphology of potato plantlets
in vitro: using light emitting diode as a light source for
micropropagation. Acta Hort 393:189194
Morini S, Trinci M, Zacchini M (1991) Effect of different photope-
riods on in vitro growth of plum rootstock. Plant Cell Tiss Org
Cult 25:141145
Mosich SK, Ball EA, Arditti J (1973) Propagation clonal de
Dendrobium por medio del cultivo de nodos. Orquidea (Mexico)
3:244260
Mosich SK, Ball EA, Arditti J (1974a) Clonal propagation of orchids
by means of node cultures. Am Orchid Soc Bull 43:10051061
Mosich SK, Ball EA, Flick BH, Arditti J (1974b) Klonvermehrung
von Dendrobium durch die Kultivierung von Stammnodien.
Orchidee 25:129134
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue. Physiol Plant 15:473496
Nanya K, Ishigami Y, Hikosaka S, Goto E (2012) Effects of blue and
red light on stem elongation and flowering of tomato seedlings.
Acta Hort 956:261266
Ng CY, Saleh NM (2011) In vitro propagation of Paphiopedilum
orchid through formation of protocorm-like bodies. Plant Cell
Tiss Org Cult 105:193202
Nhut DT, Takamura T, Watanabe H, Murakami A, Murakami K,
Tanaka M (2002) Sugar-free micropropagation of Eucalyptus
citriodora using light-emitting diode (LEDs) and film-rockwool
culture system. Environ Control Biol 40:147155
Nhut DT, Takamura T, Watanabe H, Okamoto K, Tanaka M (2003)
Responses of strawberry plantlets cultured in vitro under
superbright red and blue light-emitting diodes (LEDs). Plant
Cell Tiss Org Cult 73:4352
Nhut DT, Takamura T, Watanabe H, Okamoto K, Tanaka M (2005a)
Artificial light source using light-emitting diodes (LEDs) in the
efficient micropropagation of Spathiphyllum plantlets. Acta Hort
692:137142
123
Acta Physiol Plant (2015) 37:136
Nhut DT, Trang PTT, Vu NH, Thuy DTT, Khiem DV, Van Tran
Thanh K (2005b) A wounding method and liquid culture in
Paphiopedilum delenatii propagation. Prop Ornam Plant
5(3):156161
Nhut DT, Thuy DTT, Don NT, Luan VQ, Hai NT, Van Tran Thanh K,
Chinnappa CC (2007) Stem elongation of Paphiopedilum
delenatii Guillaumin and shoot regeneration via stem node
culture. Prop Ornam Plant 7(1):2936
Noiton D, Vine JH, Mullins MG (1992) Effects of serial subculture
in vitro on the endogenous levels of indole-3-acetic acid and
abscisic acid and rootability in micro-cuttings of Jonathan
apple. Plant Growth Regul 11:377383
Nuraini I, Shaib MJ (1992) Micropropagation of orchids using scape
nodes as the explant material. Acta Hortic 292:169172
Parker MW, Hendricks SB, Borthwick HA, Wxent FW (1949)
Spectral sensitivity for leaf and stem growth of etiolated pea
seedlings and their similarity to action spectra for photoperi-
odism. Amer J Bot 36:194204
Parks BM, Folta KM, Spalding EP (2001) Photocontrol of stem
growth. Curr Opin Plant Biol 4:436440
Patcharawadee W, Eric B, Kongkanda C, Sureeya T (2011) Effect of
cytokinins (BAP and TDZ) and auxin (2,4-D) on growth and
development of Paphiopedilum callosum. Kasetsart J Nat Sci
45:1219
Platten JD, Foo E, Elliott RC, Hecht V, Reid JB, Weller JL (2005)
Cryptochrome 1 contributes to blue-light sensing in pea. Plant
Physiol 139:14721482
Poudel PR, Kataoko I, Mochioka R (2008) Effect of red-and blue-
light-emitting diodes on growth and morphogenesis of grapes.
Plant Cell Tiss Org Cult 92:147153
Rajapakse NC, Pollock RK, McMahon MJ (1992) Interpretation of
light quality measurements and plant response in spectral filter
research. J Hort Sci 27:12081211
Ramage CM, Williams RR (2001) Mineral nutrition and plant
morphogenesis. In Vitro Cell Dev Biol-Plant 38:116124
Rotor G (1949) A method of vegetative propagation of Phalaenopsis
species and hybrids. Am Orchid Soc Bull 18:738739
Sandal I, Bhattacharya A, Ahuja PS (2001) An efficient liquid culture
system for tea shoot proliferation. Plant Cell Tiss Org Cult
65:7580
Schaefer S, Medeiro SA, Ramrez JA, Galagovsky LR, Pereira-Netto
AB (2002) Brassinosteroid-driven enhancement of the in vitro
multiplication rate for the marubakaido apple rootstock [Malus
prunifolia (Willd.) Borkh]. Plant Cell Rep 20:10931097
Schenk RU, Hildebrandt AC (1972) Medium and techniques for
induction and growth of monocotyledonous and dicotyle-donous
plant cell cultures. Can J Bot 50:199204
Sim GE, Loh CS, Goh CJ (2007) High frequency early in vitro
flowering of Dendrobium Madame Thong-In (Orchidaceae).
Plant Cell Rep 26:383393
Smith MAL, Spomer LA (1994) Vessels, gels, liquid media and
support systems. In: Aiktken-Christie J, Kosai T, Smith MAL
(eds) Automation and environmental control in plant tissue
culture. Kluwer Academic Publishers, Dordrecht, pp 371404
Sorce C, Picciarelli P, Calistri G, Lercari B, Ceccarelli N (2008) The
involvement of indole-3-acetic acid in the control of stem
elongation in dark- and light-grown pea (Pisum sativum)
seedlings. J Plant Physiol 165(5):482489
Stewart J, Button J (1975) Tissue culture studies in Paphiopedilum.
Amer Orchid Soc Bull 35:8895
Tanaka M, Takamura T, Watanabe H, Endo M, Yanagi T, Okamoto K
(1998) In vitro growth of Cymbidium plantlets cultured under
super bright and blue light-emitting diodes (LEDs). J Hort Sci
Biotech 73:3944
Teob ES (1989) Orchids of Asia. Times Books International,
Singapore, p 317
Acta Physiol Plant (2015) 37:136
Tibbitts TW, Morgan DC, Warrington JJ (1983) Growth of lettuce,
spinach, mustard and wheat plants under four combinations of
high-pressure sodium, metal halide and tungsten halogen lamps
at equal PPFD. J Amer Hort Sci 108:622630
Tomoki H, Tadashi M, Kazuhiro Y, Yuko Y, Keimei O (2014)
Synthesis of new brassinosteroid biosynthesis inhibitor with
coumarin moiety as a fluorescent probe. Inter J Chem Eng App
5(5):379383
Udomdee W, Wen PJ, Chin SW, Chen FC (2012) Shoot multiplica-
tion of Paphiopedilum orchid through in vitro cutting methods.
Afr J Biotech 11(76):1407714082
Urata U, Iwanaga ET (1965) The use of Ito-type vials for vegetative
propagation of Phalaenopsis. Am Orchid Soc Bull 35:410413
Vacin E, Went F (1949) Some pH changes in nutrient solution. Bot
Gar Conser New 110:605613
Page 11 of 11 136
Wang Q, Tang H, Quan Y, Zhou G (1994) Phenol induced browning
and establishment of shoot tip explants of Fuji apple and
Jinhua pear cultured in vitro. J Hortic Sci 69:833839
Wongnok A, Piluek C, Techasilpitak T, Tantivivat S (2008) Effects of
light emitting diodes on micropropagation of Phalaenopsis
orchids. Acta Hort 788:149156
Yepes LM, Aldwinckle HS (1994) Micropropagation of thirteen
Malus cultivars and rootstocks, and effect of antibiotics on
proliferation. Plant Growth Regul 15:5567
Ziv M (1989) Enhanced shoot and cormlet proliferation in liquid
cultured gladiolus buds by growth retardants. Plant Cell Tiss Org
Cult 17:101110
Ziv M, Halevy AH (1983) Control of oxidative browning and in vitro
propagation of Strelitzia reginae. HortScience 18:10851087
123